Macrophages and microglial cells are the major reservoirs of HIV-1 in the brain. Although the cells actively produce HIV-1, mature virus particles bud mostly into intracellular vesicles rather than into the extracellular space, as in T lymphocytes. Maintenance of this intracellular virus cache in the brain may constitute a major impediment to its eradication and to amelioration of neurological disease. We hypothesize that the pathway of Gag-Gag protein interactions during HIV-1 particle assembly distinguishes virus replication in macrophage from that in other cell types. We have previously constructed several systems for the analysis of HIV-1 protein interaction and particle assembly and propose to apply these mutagenesis and biochemical strategies to delineate the course of virus particle formation in macrophages and microglial cells. Our Specific Aims are 1) To map the pathway of virion assembly in specific cellular compartments in macrophages. 2) To identify the Gag domains mediating the formation of specific assembly intermediates in macrophages. 3) To determine the kinetics of HIV assembly in macrophages. 4) To identify the cryptic Golgi/ER targeting signal in HIV-1 Gag that determines intracellular localization of virions in macrophages. These Aims will be accomplished by infection of primary macrophages and microglial cells or transformed cell lines with HIV or by transduction of specific expression vectors followed by electron and immunofluorescence microscopy or by cellular fractionation and protein analysis. Our studies will identify the specific viral determinants and assembly intermediates distinguishing HIV particle formation and budding in macrophages and and microglial cells and thus provide the molecular basis for novel antiviral therapies to eliminate this central reservoir of HIV-1 in the infected patient.